Pathogen detection using aptamer molecular beacons using a mobile device

ABSTRACT

This disclosure pertains to a testing method for a target pathogen. The method uses biosensors with particular fluorescence characteristics, such that when the biosensor binds to a target pathogen, a fluorophore may emit light if excited. The biosensor may be an aptamer-based biosensor with a fluorophore reporter and a quencher. The excitation of the fluorophore and the detection of fluorescence may be made through the use of a flashlight source and a camera from a mobile device, such as a smartphone.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application claims priority of U.S. provisionalpatent application Ser. No. 63/044,602 filed on 26 Jun. 2020.

TECHNICAL FIELD

The field of this invention is the detection of pathogens usingaptamers, particularly by using Aptamer Molecular Beacons (AMB) and acamera-equipped device, such as a mobile device, smartphone orequivalent.

BACKGROUND

Aptamers are oligonucleotide or peptide molecules that bind to aspecific target molecule and that have been studied as early as 1990.Aptamer-based biosensors have been developed for use in a wide varietyof sensing technique, such as electrochemical, optical andmass-sensitive analytical techniques. Aptamers exhibit many advantagesas recognition elements in biosensing when compared to traditionalantibodies. They are small, chemically stable and cost effective. Moreimportantly, aptamers offer remarkable flexibility and convenience inthe design of their structures, which has led to novel biosensors thathave exhibited high sensitivity and selectivity.

Aptamers are well known to have distinctly different conformations andstructures before and after binding with the targets. Influorophore-based aptamers, the structural changes of aptamer may modifythe structure such that a quencher-reporter structure (i.e. an aptamermolecular beacon) may be separated, thus allowing the reporter toexhibit fluorescence when excited with a light source at a certainwavelength.

Aptamers may be developed and selected specifically for a targetpathogen and therefore may be used in diagnostic tests for almost anyinfection. The fluorescence produced by an aptamer molecularfluorescence beacon is generally detected through fluorescencespectroscopy or fluorometry, which are costly equipment which are notnecessarily portable. Additionally, the complexity of operations of suchequipment is inherently high and require specific training. Therefore,aptamer-based testing is not accessible for most applications and maynot be used in most facilities.

Since aptamers may be developed for a specific pathogen, its diagnostictesting capabilities are incredible, but the current limitations foranalyzing the aptamer's response do not allow for its widespread use. Assuch, it would be beneficial to have an aptamer-based testing devicewhich may be affordable, operable by almost anyone and with no highlyspecialized devices (other than the aptamer filled testing vial). Aportable testing unit may further be desirable as it may allow testinganywhere.

SUMMARY

Applicant has discovered a testing method for a target pathogen that maybe used with a mobile device, such as a smartphone, instead ofspecialized devices. By selecting an Aptamer that binds uniquely to thevirus that is the target for a test, and by attaching a selectedappropriate fluorophore and matching quencher for the chosen aptamer, anAptamer Molecular Beacon may be formed. This Aptamer Molecular Beacondisplays the property that prior to binding to the specific virus, ithas a shape where the quencher is in proximity to the fluorophore, andwhen it binds, the shape changes and the quencher molecule separatesfrom the fluorophore. The fluorophore then, on receiving incident lightwill fluoresce at a different wavelength than the incident light. Theaptamer, the fluorophore and the quencher can be specifically selectedto have an excitation wavelength that approximately is the same as asmartphone LED's spectral peak and the emitted light can be within therange of sensitivity of the smartphone's camera sensors.

As a matter of fact, most mobile devices use the same type of LED flashtechnology which produces white light by using a blue GaN light-emittingdiode (LED) (e.g. emitting with a peak at about 460 nm) with a layer oflight-emitting phosphor to produce other wavelengths so that thecombination of wavelengths appears to be white to the human eye. Thiswhite LED output contains a reasonably narrowband source of opticalpower in the blue part of the spectrum

A first broad aspect is an aptamer molecular beacon testing device fordetecting a pathogen including: a receptacle for receiving a test vial;a coupler for receiving a mobile device having a camera and a flashlight source, the coupler being positioned with respect to thereceptacle to allow for light from the flash light source to reach thetest vial and for fluorescence light from the test vial generated inresponse to the light from the flash light source to reach the camera;and a lightproof enclosure for preventing external ambient light fromreaching the camera when the mobile device is coupled.

In some embodiments, the aptamer molecular beacon testing device furtherincludes a bandpass optical filter to allow the light from the flashlight source to reach the Aptamer Molecular Beacon while allowing thefluorescence from the fluorophore bound to the aptamer to reach thecamera.

In some embodiments, the mobile device and the test vial are completelyenclosed in the lightproof enclosure.

In some embodiments, the coupler is a slot in the lightproof enclosure.

In some embodiments, the coupler includes an opaque seal.

In some embodiments, the lightproof enclosure further includes a movablegate to access the test vial receptacle, the movable gate beingconfigured to prevent the ambient light from leaking inside thelightproof enclosure in a closed position.

In some embodiments, the coupler is one of a mobile device cover or anattachable mobile camera cover.

In some embodiments, the aptamer molecular beacon testing device furtherincludes a reference element operable to emit light in response toreceiving a light excitation.

In some embodiments, the test vial receptacle further includes a movablepart operable to displace the reference element from a rest position toa deployed position when a replaceable test vial is fully inserted inthe test vial receptacle, the reference element being outside afield-of-view of the camera in the rest position and inside thefield-of-view in the deployed position.

In some embodiments, the aptamer molecular beacon testing device furtherincludes at least one of a mobile device adapter and an apertureselector configured to adapt the lightproof enclosure to differentmobile devices.

In some embodiments, the bandpass optical filter includes a gapconfigured to allow any light source through.

A second broad aspect is an aptamer molecular beacon testing device fordetecting a pathogen including: a lightproof enclosure configured toreceive a test vial, the lightproof enclosure including at least a lightsource and a camera cell.

In some embodiments, the aptamer molecular beacon testing device furtherincludes a communication module operable to transmit information to acomputing device and receive information from the computing device.

In some embodiments, the aptamer molecular beacon testing device furtherincludes a battery providing power to at least the light source and thecamera cell.

A third broad aspect is a pathogen testing system including: an aptamermolecular beacon testing device; and a replaceable aptamer molecularbeacon test vial configured to be placed in the test vial receptacle,the replaceable aptamer molecular beacon test vial having a body beingconfigured to receive a sample fluid from a tested subject, the testvial body including a solution of aptamer molecular beacons and acarrier liquid medium, wherein the aptamer molecular beacons areselected to bind to a protein, RNA or DNA of a target pathogen andfurther include a reporter molecule that is capable of receiving lightand emitting light after binding to the target pathogen.

In some embodiments, the lightproof enclosure is configured to positionthe mobile device and the replaceable aptamer molecular beacon test vialsuch that a light from the flash light source illuminates thereplaceable aptamer molecular beacon test vial and such that the camerais operable to capture the emitted light from the Aptamer MolecularBeacons.

In some embodiments, the reporter molecule emits light at a wavelengthwhich corresponds to a peak sensitivity of at least one light sensor ofthe mobile device.

In some embodiments, an optical filter is configured to cover a lens ofthe camera while leaving the flash light source uncovered to limit alight received by the camera to a wavelength corresponding to anemission wavelength of the reporter molecule. In such a case,simultaneous excitation of the fluorophore and detection of the emissionlight can be possible. This is useful when the lifetime of thefluorescence is very short. This can also be useful for eliminatingbackground light when ambient light is otherwise able to reach thecamera.

In some embodiments, the replaceable aptamer molecular beacon test vialincludes an opaque seal surrounding a removable cap configured to beattached to the test vial.

In some embodiments, the replaceable aptamer molecular beacon test vialis a test slat, the container being a top surface of the test slat.

In some embodiments, the pathogen testing system further includes areflective sticker over a surface of the container.

In some embodiments, the reflective sticker includes a barcode.

A fourth broad aspect is a pathogen testing aptamer molecular beacontest vial configured to be placed over a camera and a flash light sourceof a mobile device, including a molecular beacon aptamer test solution,wherein the Aptamer Molecular Beacons are selected to bind to a protein,RNA or DNA of a target pathogen and further include a reporter moleculethat is capable of receiving light and emitting light after binding tothe target pathogen, a reflective or opaque material covering a surfaceof the test vial and having a first window for placement over the flashlight source of the mobile device camera and a second window forplacement over a lens of the camera of the mobile device, the secondwindow having a bandpass optical filter area for absorbing at least thelight from the flash light source while allowing the fluorescence lightto reach the camera.

In some embodiments, the vial includes a lid covering a fluid chamber.

A fifth broad aspect is a mobile device readable non-transitory memorystoring instructions executable by a mobile device, including: at leastone instruction for causing a flash module of the mobile device to beoperated; at least one instruction for at least one camera of the mobiledevice to capture an image of a vial including a solution of a testsubject fluid, a quantity of Aptamer Molecular Beacons and a carrierliquid medium; at least one instruction for a processor of the mobiledevice to compute an infection status based on the captured image; andat least one instruction for the processor to display the infectionstatus on a display module of the mobile device.

In some embodiments, the memory further includes at least oneinstruction for causing operation of the flash module to be stoppedbefore executing the at least one instruction for at least one camera ofthe mobile device to capture an image.

In some embodiments, the memory further includes at least oneinstruction for storing at least one of the captured image and theinfection status in a storage module of the mobile device.

In some embodiments, the memory further includes at least oneinstruction for sending at least one of the captured image and theinfection status to a remote server.

In some embodiments, the memory further includes at least oneinstruction to scan a barcode or receive a code associated with the vialand to determine at least one of the state of a replaceable test vialand a target pathogen being tested.

In some embodiments, the memory further includes at least oneinstruction to prevent the mobile device from being operable todetermine infection status when the state of the replaceable test vialindicates an invalid replaceable test vial.

In some embodiments, the memory further includes at least oneinstruction to confirm at least one of a presence of the replaceabletest vial and a negative infection status test result by processing thecaptured image and identifying a reference element.

A sixth broad aspect is a method for detecting a pathogen including:starting a mobile device software testing application; adding a testsubject sample fluid to a replaceable aptamer molecular beacon test vialincluding a solution of Aptamer Molecular Beacons and a carrier liquidmedium, wherein the Aptamer Molecular Beacons are selected to bind to aprotein, RNA or DNA of a target pathogen and further include a reportermolecule that is capable of receiving light and emitting light afterbinding to the target pathogen; mixing a content of the replaceableaptamer molecular beacon test vial; aligning the aptamer molecularbeacon test vial with a camera and a flash light source of the mobiledevice; and performing a test to detect a presence of the targetpathogen by running the mobile device software testing application.

In some embodiments, the performing the test includes causing the flashlight source to be operated and the camera to capture an image of thereplaceable aptamer molecular beacon test vial.

In some embodiments, the method further includes at least one ofcomputing an infection status based on the captured image and displayingthe infection status on a display of the mobile device.

In some embodiments, the method further includes storing at least one ofthe captured image and an infection status in a storage module of themobile device.

In some embodiments, the method further includes sending at least one ofthe captured image and the infection status to a remote server.

In some embodiments, the method further includes scanning a barcode withthe mobile device to determine at least one of the states of areplaceable test vial and a target pathogen being tested.

In some embodiments, the method further includes preventing the mobiledevice from being operable to determine infection status when the stateof the replaceable test vial indicates an invalid replaceable test vial.

In some embodiments, the method further includes confirming at least oneof a presence of the replaceable test vial and a negative infectionstatus test result by processing the captured image and identifying areference element.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood by way of the following detaileddescription of embodiments of the invention with reference to theappended drawings, in which:

FIG. 1 is a composite illustration of a fluorescence Aptamer MolecularBeacon in a folded and attached position, as found in the prior art;

FIG. 2 is a schematic of an exemplary aptamer molecular beacon testingdevice for a mobile device;

FIG. 3A is a schematic illustrating some internal components of anexemplary aptamer molecular beacon testing device for a mobile device;

FIG. 3B is an illustration of an exemplary aptamer molecular beacontesting device setup comprising a mobile device inserted in theenclosure;

FIG. 3C is a schematic of an exemplary aptamer molecular beacon testingdevice with a gated access to the sample enclosure;

FIG. 3D is a schematic of an exemplary aptamer molecular beacon testingdevice with a sample engagement and reference unit;

FIG. 3E is a schematic of an exemplary aptamer molecular beacon testingdevice with a removable flash and camera apertures part;

FIGS. 4A-4B are schematic drawings of an exemplary aptamer molecularbeacon testing device fixed to a mobile device;

FIG. 4C is a schematic drawing of an exemplary standalone aptamermolecular beacon testing device which includes a light source, a cameramodule and a communications module;

FIGS. 5A-5E are illustrations of exemplary aptamer molecular beacon testvials;

FIG. 6 is a graph of an exemplary fluorescent emission response to amobile device flash by an Aptamer Molecular Beacon;

FIG. 7 is a diagram of the modules of an exemplary mobile device runningan aptamer molecular beacon test; and

FIG. 8 is a flowchart of an exemplary method of testing for a targetpathogen using an aptamer molecular beacon testing device with a mobiledevice.

DETAILED DESCRIPTION

Recent advances in bioscience have made it possible to detect pathogenswithout the complex methods being employed up to now. These AptamersMolecular Beacons bind to specific pathogens that can in turn emit lightwhen being bound to the target pathogen. Devices such as smartphoneshave the computing means to analyze the results and provide theconclusion, and store the results locally or communicate them, if sodesired.

Aptamers

Aptamers are single stranded nucleic acid molecules or peptide moleculesthat fold into complex three-dimensional conformations that canselectively bind to a specific target, including proteins, peptides,carbohydrates, small molecules, toxins, and even live cells. Aptamersassume a variety of shapes due to their tendency to form helices andsingle-stranded loops. Aptamers can be developed to bind to any desiredtarget. The aptamer can be generated via in-vitro selection or via SELEX(systematic evolution of ligands by exponential enrichment) ranging frommetal ions to cells. On the molecular level, the aptamers will bind toits cognate target by non-covalent interactions. As such, aptamers maybe specifically designed to bind to any pathogens (e.g. viruses,bacteria, spores, etc.) to help in diagnosis or to deliver drugs tospecific cells inside a patient. One such aptamer has been proven tobind to SARS-CoV-2 S1 spike protein on the surface of the virus. OtherAptamers can be chosen or designed to bind to other proteins or RNA orDNA of other viruses and pathogens.

Fluorophores

Fluorophores are molecules that can be very small, <1200 Daltons thatmay be attached to one end of the aptamer. One such fluorophore thatexhibits the desired properties that match the characteristics of thesmartphone LED and cameras is Atto 465 (e.g. from Sigma Aldrich). Asdescribed herein, the fluorophore may be used as the reporter in anAptamer Molecular Beacon.

Quencher

A quencher is also a small molecule that can be attached to the oppositeend of the aptamer than the fluorophore. The quencher, while inproximity with the fluorophore, absorbs the energy from the fluorophorethat would otherwise be emitted. On separation from the fluorophore, thequencher stops being operable to absorb this energy, therefore allowingthe fluorophore to emit light. This separation occurs, in anappropriately designed aptamer, on binding to the target protein, RNA orDNA. One quencher that will attach and quench the Atto 465 fluorophoreis the Iowa Black® Fluorescence Quencher.

Aptamer Molecular Beacons (AMB)

As illustrated in FIG. 1, a Molecular Beacon may be an aptamer with thefluorophore, which is the AMB reporter, and quencher attached at the twoend of the aptamer held in a hairpin-loop conformation (around 20 to 25nucleotides) by complementary stem sequences (around 4 to 6 nt) at bothends of the probe. The 5′ and 3′ ends of the probe contain a reporterand a quencher molecule, respectively. The loop may be a single-strandedsequence complementary to the target sequence. The proximity of thereporter and Quencher causes the quenching of the natural fluorescenceemission of the reporter.

Aptamer Molecular Beacons hybridize to their specific target sequencecausing the hairpin-loop structure to open and separate the reporter andthe quencher. As the Quencher is no longer in proximity to the reporter,fluorescence emission may take place when the necessary excitation isprovided to the molecule. Therefore, the measured fluorescence signalmay be directly proportional to the amount of target pathogen. As amatter of fact, the more testing solution contains the target pathogen,the more Aptamer Molecular Beacons will be attached and have theirreporter separated from their quencher (i.e. the fluorescence signalwill be stronger). There are different 3D shapes and changes to themorphology of the Aptamer Molecular Beacons on binding to follow thechange on binding. Similarly, the fluorophore and quencher conjugationto the aptamer may be different. In some cases, the quencher can bereleased.

As is known in the art, the fluorophore may be selected to emit lightfor a particular wavelength of absorbed light.

Aptamer Molecular Beacon (AMB) Testing Device

As described herein, the Applicant has discovered a method of testingfor a pathogen using a mobile device and an Aptamer Molecular Beaconsolution. As a matter of fact, it is possible to select an AMB with afluorophore excitable by the main wavelength of the light emitted by amobile device's flash. Given the optical properties of most camerasincluded in current mobile devices, the camera may be operable to detectfluorescence emitted by AMBs bound to target pathogens. In order todetect low levels of emitted fluorescence, a bandpass filter may berequired. The filter may thus allow the mobile device's camera to sensea small amount of the target pathogen, such as would be present in apatient in early stages of infection while further filtering outfluorescence from water in the solution and other contaminants in thesaliva such as food particles and fruit particles.

In some embodiments, the bandpass filter may not be required for thesystem to properly function. As such, the bandpass filter may not berequired when a sufficiently lightproof enclosure (i.e. no leakage ofambient light) and a fluorophore operable to emit light for a longenough period after the excitation light source has stopped are used. Asa matter of fact, these may allow the testing device to flash the lightsource and to capture an image after the light source has beenextinguished (i.e. the only light being captured would be the lightemitted by the fluorophores, therefore no filter may be required).

In some embodiments, there AMB may be selected to bind with a givensurface protein of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2, the virus causing COVID-19) and where the quencher and thereporter are chosen to be excited by a light source having approximately450 nm and emit at 490 nm, or appreciably similar wavelength. Someoneskilled in the art will appreciate the fact that the AMB may be selectedto bind to any pathogen.

In order to perform a diagnostic test to determine the infectious stateof a person with regards to a target pathogen, a sample of the saliva ornasal mucus may be taken with a swab and inserted into a vial. The vialcontains the AMB and a carrier liquid medium, such that when the vial isshaken the person's saliva or nasal mucus may mix with the AMB andcarrier liquid medium.

Reference is now made to FIG. 2, which is a schematic of an exemplaryaptamer molecular beacon testing device for a mobile device. In thisembodiment, a fixture 29, which may be a lightproof enclosure, containsa slot for a mobile device 27 (e.g. smartphone, tablet, etc.) and areceptacle for a test vial 25. The receptacle for the test vial 25 maybe a slot and may be positioned inside the fixture 29 such that thedistance from the vial to the flash of the smartphone and the distancebetween the vial and the camera lens selected to gather the lightemitted by the reporter is constant within a small covariance. Thisensures that the Beer-Lambert law calculations in the mobile deviceapplication may be respected. Someone skilled in the art will appreciatethat, as such, the extinction ratio of the medium is known, the distancefrom the flash is known and the light flux is known.

In some embodiments, the slot for the mobile device 27 may be a couplerwhich effectively joins the mobile device to the fixture 29. Forexample, a relatively small fixture 29 may be positioned over a mobiledevice, such that it only covers an area over a camera and a flash ofthe mobile device. In such example, the slot for the mobile device 27may be replaced by the connection between the mobile device and thefixture 29, which is herein defined as a coupler. The coupler may belightproof, such that no ambient light may leak to the inside of theenclosure/fixture 29. In some embodiments, the coupler may be a mobiledevice cover that fits over the mobile device and holds the other partsof the testing device in the required position over the flash andcamera(s).

FIG. 3A illustrates internal components of an exemplary aptamermolecular beacon testing device for a mobile device. In this embodiment,a mobile device 39 (e.g. smartphone, tablet, etc.) may be insertedinside the fixture's 29 slot for the mobile device. As the mobile device39 is positioned correctly inside the slot, its flash 33 and camera 37may be aligned with their associated apertures in the fixture 29 andthereafter also aligned with the test vial 31. The test vial 31 may beinserted in the fixture's 29 slot for the test vial, such that the vialis correctly positioned to allow its content to be illuminated by theflash 33 and for its potential fluorescence to be detected by the camera37.

The mobile device's 39 camera 37 may be aligned with a filter 35, whichmay be a notch optical filter specific to 490 nm (e.g. a bandpass filterwith a narrow band around 490 nm). As the mobile device's 39 may have aflash 33 made of Light Emitting Diodes that have a fundamental lightemission between 450 to 460 nm, the fluorophore may emit light at around490 nm such that it may be the only light to pass-through the filter 35.In some embodiments, the optical filter may include a small gap to allowany light to passthrough, such that an aligned camera may sense theintensity of flash and may be able to confirm the presence of a vialinserted in the vial's receptacle. In some embodiments where the filteris not required, as described herein, the detection of the vial may bedone by capturing a first image of the vial while the light source isoperating. The testing may subsequently be done by operating once morethe light source and thereafter capturing an image once the light sourcehas extinguished (such that only a presence of fluorophores may beemitting). Someone skilled in the art will appreciate that it may befeasible to continuously capture a series of image, some of them beingwhile the light source is operating (i.e. confirming the presence of thevial) and some of them after the light source has extinguished.

The reporter quantum efficiency and the absorbance of the light by theliquid medium will determine the light emission quantity in number ofphotons. As mobile devices 39 have different of cameras and that somemobile devices may have multiple camera sensors, the testing system,through the control application, may select the camera lens with thehighest quantum efficiency for the testing, this being about 75% at 490nm. Additionally, any image processing capabilities of smartphones orother mobile devices may be used to enhance the image captured duringthe test. These capabilities may include post-processing software, whichmay include artificial intelligence image processing. This may enhancethe sensitivity of the test and therefore allow for detection of thetarget pathogen in relatively small quantities.

The sensors in the smartphone or similar device can use a single cell ofthe camera sensor or a plurality, or all, the cells of the sensor tocalculate the results of the test performed. This maybe done immediatelyas part of the test process or stored over time and multiple tests andthen analyzed. The fluorophore emission light can be taken from aspecific region of interest within the acquired image. Spatial intensityanalysis can be used to confirm that the intensity observed matchesintensity from the expected distribution of the aptamer within thesolution of the vial.

It will be appreciated that the test result can apply a simple thresholdto the total amount of light observed to give essentially a binaryresult, or the amount of light received can be used to qualify orquantify the result. This can allow the positive or negative result tobe qualified with a confidence value that health care professionals canconsult. In some tests, the amount of light in the acquired image can beused to be correlated with a disease state or disease severity.

The fluorophore can have a lifetime that will permit the emission lightto be captured by the mobile device's camera once the flash light sourceis turned off.

A reflector may further be placed behind the vial to reflect any lightemitted by the fluorophore back to the camera, similarly a tube can beplaced between the vial and the camera to eliminate any external lightfrom effecting the reading. A similar absence of ambient or externallight maybe achieved by another means as well.

FIG. 3B illustrates an exemplary aptamer molecular beacon testing devicesetup comprising a mobile device 39 inserted in the enclosure. In thisembodiment, the fixture 29 may have a long form factor such that thetesting device may be placed on a mostly horizontal surface (e.g. atable, counter, desk, etc.) without tilting over or moving throughoutthe test. As a matter of fact, a smaller form factor may be prone totilting or moving once the mobile device 39 is inserted at one end ofthe device. It will be appreciated that any different form factor may beused for the testing device as it may be specified to hold the devicethroughout the test in the accompanying testing method description.

In the embodiment of FIG. 3B, a test vial slot 25 in the fixture 29 isincluded on the opposite side of the mobile device's 39 slot. The testvial slot 25 may be aligned with the mobile device's 39 camera(s) andflash. As such, the test vial slot 25 may be positioned differentlydepending on the model of mobile device 39 to be used in the testingdevice. The test vial slot 25 may be a cavity which may be completelycovered by an opaque vial cap, such that no ambient light may enterinside the fixture's 29 testing chamber.

FIG. 3C is a schematic of an exemplary aptamer molecular beacon testingdevice with a gated access to the sample enclosure. In this embodiment,the mobile device slot 27 may have an opaque seal 41 enclosing itsopening in the fixture 29, such that no ambient light may leak throughthe opening into the testing chamber. Ambient light leakage mayotherwise impact the mobile device's camera(s) sensors to a point wheretheir sensibility to the fluorescent light that may be emitted by atested sample in a test vial is detrimentally affected.

In the embodiment of FIG. 3C, there may be a gate 43 providing an easyaccess to the test vial slot 25. Similar to the opaque seal 41surrounding the mobile device slot 27, an opaque seal 41 may surroundthe gated access point. As such, when the gate 43 is closed and pressedagainst the fixture 29, the opaque seal 41 may prevent any ambient lightfrom leaking inside the testing device.

Now referring to FIG. 3D, which is a schematic of an exemplary aptamermolecular beacon testing device with a sample engagement and referenceunit. In some embodiments, it may be desirable to include a system thatmay indicate whether the test vial is correctly and completely insertedinside the test vial slot 25. This may be necessary to ensure that anopaque seal is properly formed by a test vial cap (i.e. no ambient lightmay leak and impact test results) or that all of the part in which thevial is filled by the mix of AMB, carrier liquid medium and sample fromthe test subject is exposed to the light source (e.g. flash).Additionally, this system may be used as a reference point to calibratethe mobile device's camera(s) and/or to show that a negative test hasbeen successfully completed and is not the result of an error ormalfunction.

For example, FIG. 3D illustrates a mechanical engagement and referencesystem in which the complete insertion of a test vial inside the testvial slot 25 pushes an engagement member 45 towards a bottom of thefixture 29. The engagement member 45 may thereafter push a rotary member49 on which is fixed a fluorescent part 51. The engagement member 45 maybe connected to a biasing member 47 (e.g. a compression spring) whichmay reset the engagement and reference system to a resting position oncea test vial is removed from the test vial slot 25.

In its resting position, the fluorescent part 51 of the engagement andreference system may be outside of the field-of-view of the mobiledevice's camera(s). When a test vial is fully engaged and the engagementand reference system is in its deployed position, the fluorescent part51 may be inside the field-of-view (FoV) of the mobile device'scamera(s). The deployed position of the engagement and reference systemmay be positioned such that it does not interfere with the detection ofAMBs from the test vial while remaining inside the FoV of the camera(s).This may be particularly useful to ensure that no malfunction happenedduring a test, mostly for a negative test result.

As a matter of fact, a negative test result necessarily means that noAMBs emitted light, or that an insignificant number of AMBs emittedlight. Thus, a negative result for a test may be the detection of acompletely dark testing chamber inside the testing device. Some deviceor mobile device application failures or malfunction may lead to similarresults, such as the non-operation of the flash, an issue with the flashand/or camera controls, etc. This may mean that a false-negative resultmay be given to the person being tested, potentially leading to theperson unknowingly spreading the disease.

When equipped with an engagement and reference system such as the onedescribed herein, the fluorescent part 51 may be present in the sensedpicture. Thus, while there may be no light emitted from the test vial(i.e. negative test result), the test may be confirmed as havingsuccessfully taken place by the recording of the fluorescent part 51being in the FoV.

Someone skilled in the art will appreciate that any other configurationof an engagement and reference system may be used without departing fromthe teachings of this disclosure. Systems may also be used for only oneof the functions of the described engagement and reference system (e.g.only for engagement of the vial or only for the reference). As such,other mechanical configurations or other systems (e.g. electrical,magnetic, etc.) may be used in lieu of the one described herein. Opticalengagement and reference system may use other light sources (e.g.phosphorescent). A static reference system may be used, in which afluorescent part 51 or equivalent is fixed at the “deployed” position(i.e. always in the FoV of the camera(s)).

Now referring to FIG. 3E, which is a schematic of an exemplary aptamermolecular beacon testing device with a removable flash and cameraapertures part 52. As there are a significant number of mobile deviceson the market, it may be desirable to have a modular AMB testing devicefixture 29 design. The mobile device slot may be adapted to fit mostsmartphones (most tablets in a bigger fixture, etc.). In order to ensurethe position of the mobile device inside the slot, such that the flashand camera are correctly positioned and do not move during the test, thedevice may include a replaceable mobile device adapter 50 configured tofit around the mobile device and resting on the sides of the mobiledevice slot. The testing device may thus have a mobile device adapter 50specific for the mobile device being used for the test.

Similarly, as the size and the positioning of the flash and camera(s)differs between mobile device models (and its position inside the mobiledevice slot), a replaceable aperture selector 52 may be inserted in thefixture 29 in an aperture selector slot that may be positioned betweenthe mobile device slot and the test vial slot 25. The aperture selector52 may include the necessary flash aperture 33′ and the one or morecamera apertures 37′ that are associated with the mobile device to beused and inserted inside the mobile device slot. Additionally, theopening of the aperture selector slot on the fixture 29 may include anopaque seal surrounding the area.

Using different mobile device adapters 50 and their associated apertureselector 52 may allow the user of the testing system to operate it withmultiple different devices. This may be particularly useful forbusinesses in which different operators may perform the tests and maynot necessarily use the same mobile device.

FIGS. 4A-4B illustrates exemplary embodiments of an aptamer molecularbeacon testing device directly fixed to a mobile device. As such, thetesting device fixture 29 may be attached to the mobile device 39 with aclamp 53. The fixture 29 may be further attached to the mobile device 39through a specifically purposed case (e.g. phone case, tablet case,etc.). In order to retain the smallest form factor possible, the testvial slot of the fixture 29 may not necessarily be aligned with theflash 33 and the camera 37 of the mobile device 39. As such, a samplevial 31 inserted in the test vial slot may be aligned with the camera 37and the camera aperture 37′ only. In such embodiments, the fixture 29may include a mirror 55 to redirect the light source from the flash 33,through the flash aperture 33′, towards the sample vial 31. As furtherdescribed herein, the fixture 29 may include a filter 35 between thesample vial 31 and the camera 37. Additional optics to capture morelight from the vial may be added to the system without departing fromthe teachings of this disclosure. As such, the system may includemirrors, lenses, filter for the light source and any other opticaldevice that may improve or change some optical characteristics insidethe enclosure, the test vial or other structure of the test system asdescribed herein.

To prevent any ambient light from degrading the test results, thefixture 29 may further include an opaque seal 41 between the fixture 29and the mobile device 39, the seal effectively surrounding the aperturesfor the camera(s) 37′ and the flash 33′.

FIG. 4C is a schematic drawing of an exemplary standalone aptamermolecular beacon testing device which includes a light source 33, acamera cell 37 and a communications module 38. While the aptamer testingdevice described herein may typically use a mobile device as anefficient device that includes numerous of the systems required for thetest, such as a light source, a camera, a processing and calculationsmean, the aptamer testing device may equivalently function without amobile device if the testing device fixture 29 includes all necessarysystem components.

As illustrated in the embodiment of FIG. 4C, the fixture 29 may be anopaque cylindrical container with sufficient place inside the containerto insert a test vial. As such, it will be understood that the shape ofthe fixture 29 may take any necessary form to accommodate a test vial. Afixture removable cap 36 may be fixed and remove from the fixture 29 toallow access to the container in order to add/remove a test vial. Thefixture 29 may include, on a surface to which the content of a test vialwould be exposed to, a light source 33 and a camera cell 37. The lightsource 33 may be a light emitting diode similar to the ones found inmobile device's flash. The camera cell 37 may be specifically sensitiveto the wavelength emitted by the fluorophore used in the aptamercontained in a test vial to be used inside such device. The light source33 and camera cell 37 may have all necessary components to function andbe controlled.

The embodiment of FIG. 4C may further include a communications module 38that may establish a wireless connection to any computing device (e.g.mobile device, computer, etc.). The communications module 38 may useWiFi protocols, Bluetooth protocols or any other protocols allowingexchange of data packets between the testing device and a computingdevice. In some embodiments, the communications module 38 may include aport such as to allow a wired communication to a computing device. Insome embodiments, the communications module 38 may allow a softwareapplication running on the connected computing device to control theoperation of the light source 33 and of the camera cell 37. As such, thecomputing device may trigger the light source 33 for a given time andthereafter trigger the capture of the image of the test vial to detectany light emissions from the fluorophores.

Someone skilled in the art will appreciate that other means oftriggering the different testing sequence may be used. For example, atest button may be included on the fixture 29 or on the fixtureremovable cap 36 to trigger the test. The captured image may beautomatically transferred to a paired computing device to which thecommunication module 38 is connected to. The captured image may furtherbe recorded on an on-board non-transitory memory unit, which may allowfor subsequent transfer to a computing device.

The fixture 29 may further include a power source to provide sufficientpower for the operation of the light source 33, the camera 37 and thecommunications module 38. For example, the power source may be arechargeable battery, which may be recharged by connecting a power cableto a port included on the fixture 29. In some embodiments, the batterymay be a replaceable battery in a compartment easily accessed.

Although described as communicating with a computing device to processthe captured image data and assess the infection state of the testedperson, it will be appreciated that the standalone testing device aspresented in FIG. 4C may further include necessary electronic components(e.g. microprocessor, memory (transitory and non-transitory), etc.) tointernally process all data and provide the infection state feedbackwithout relying on a connected computing device. For example, thetesting device may process the captured image data and light a greenlight or a red light depending on the determined infection state of thetested patient.

It will be further appreciated that any additional optics elements (i.e.filters, mirrors, lenses, etc.) may be added inside the fixture 29 or tothe test vial to increase or change certain optical characteristics ofthe testing device and improve its efficiency at determining theinfection state of a tested subject.

Now referring to FIGS. 5A-5D, which are illustrations of exemplaryaptamer molecular beacon test vials. AMB testing vials may have anyshape that can fit inside a testing device's test vial slot. Asdescribed herein, the testing vial may contain a mix of AMBs and carrierliquid medium and may be opened to add the tested person sample fluid(e.g. saliva, nasal mucus, etc.). FIG. 5A illustrates an embodiment inwhich a bullet-shaped vial has a container 59 and a cap 57. The vial cap57 may be twisted or pulled to be removed from the container 59, suchthat a sample fluid may be added to the mix contained in the container59. The container 59 may be transparent such as to allow light topass-through.

FIG. 5B illustrates an embodiment of a test vial in which the cap 57includes an opaque seal 61. The opaque seal 61 may fit around thetesting device's test vial slot, such that no light may leak through theopening to degrade the test results. Part of the cap 57 may extentupwards from the opaque seal 61 such that it may be manually insertedand removed from the testing device.

FIG. 5C shows an embodiment in which part of the test vial's container59 is covered by a barcode sticker 63. The barcode sticker 63 may bescanned by the mobile device being used for the testing and may ensurethat the testing vial used is an official vial for the target pathogenbeing tested (i.e. quality control, it contains the relevant AMBs and insufficient quantity, etc.). It may also be particularly useful to scan abarcode to ensure that a test vial is not re-used by the personperforming the test. Additionally, the barcode sticker 63 may have areflective underlayer, such that when the test vial is positioned withthe barcode sticker 63 facing away from the mobile device, the light maybe reflected by its reflective underlayer. This may increase thesensitivity of the test and may replace the addition of a mirror insidethe fixture.

In other embodiments, the barcode sticker 63 may be on the cap 57 or maybe on a package containing one or more test vials. In yet anotherembodiment, the barcode sticker 63 may be replaced by any other means ofconfirming the source of the test vial, such as an RFID tag. In someembodiments, the sticker may strictly be a reflective sticker added toincrease the sensitivity of the test.

FIG. 5D illustrates a different type of test sample support. The testvial may thus be replaced by a testing slide or substrate 62 over whichthe mix of AMBs, carrier liquid medium and fluid from the tested subjectmay be spread. Someone skilled in the art will appreciate that any othershape may be used as a test “vial” and that the term “vial” should notbe interpreted as limiting the scope of the present disclosure to acontainer-type test sample.

The embodiment illustrated in FIG. 5E may be used without any fixture 29as a testing device. As such, the vial's container 59, which may begenerally of rectangular shape, may include the necessary structures toallow the test. For example, the container 59 may have a reflectivesticker 64 wrapped around all but one surface and a top of thecontainer. No matter the shape of the vial, the arrangement of a flatside for optically coupling with the mobile device can be desirable. Thereflective sticker 64 may thus prevent ambient light from passingthrough the sticker 64 into the container. The removable cap may be madeout of opaque material such that, once placed on top of the vial'scontainer 59, it may overlap with the sticker 64 to further preventambient light leaking inside the container 59. In some embodiments, thereflective sticker 64 may cover a bottom section (i.e. opposite of thecap) of the container 59. In other embodiments, the bottom section ofthe container 59 may have a bottom cap opaque structure that may provideeasier hold of the vial when performing a test.

In the embodiment of FIG. 5E, one side (A) of the vial's container 59may be configured to be directly pressed against the mobile device'sflash and camera structure. As such, this side (A) may include apass-through area 33′ for the flash light source to go through thesurface into the content of the container. Additionally, there may be afiltered area with a bandpass optical filter 35 that may be placed overthe camera(s) of a mobile device during a test.

FIG. 6 is a graph of an exemplary fluorescent emission response to amobile device flash by an Aptamer Molecular Beacon. In this embodiment,the light exciting the AMB's reporters may be around 450 nm (dottedline) and the fluorescent response produced by the AMB's reporters maybe emitted at a higher wavelength.

Someone skilled in the art will appreciate that specific wavelengthsdisclosed herein may change with different models of mobile devices, asmay the sensitivity of the method and characteristics of the fixture. Asa result of such, the Aptamer Molecular Beam specifications (e.g.fluorophore and quencher used, etc.) may also change. However, whetherany or all of these changes are made to the aptamer molecular beacontesting device, the underlying principles are the same as taught herein.

Software Application

To operate the aptamer molecular beacon testing device with a mobiledevice, a software application is required. As a matter of fact, thesoftware application is required as the test requires control over theflash, the camera, computing power and other modules of the mobiledevice.

Now referring to FIG. 7, which is a diagram of the modules of anexemplary mobile device running an aptamer molecular beacon test. In amobile device, the software application 65 may have access to numerousmodules (herein defined as all the software and hardware components ofthe mobile device required to control its associated feature). As such,the software application 65 for the aptamer molecular beacon test mayaccess and operate the processing module 67, the flash module 69, thecamera module 71, the storage module 73, the connectivity module 75 andthe display module 77.

The connectivity module can establish a connection to a serverassociated with the provider of the test vials. The application 65 canbe configured to perform a test only when the user of the application 65enters a valid code associated with the test vials, such as by acquiringan image of a barcode or by entering a code found on the vial or itspackaging. The code can be validated locally by the application 65 usingconventional decryption, hash value or checksum verification, or it canbe validated by the server. If the code is invalid, the test vial shouldnot be trusted. If ever a batch of vials were to be found to bedefective, the server can report to the application 65 that the vial,even with a valid code, is defective and notify the user to seek areplacement using the display module 77.

The software application 65 may thus provide the processing module 67(which may include the mobile device's processor, transitory andnon-transitory memory units, etc.) with instructions which, when theyare executed are operable to control the other modules of the mobiledevice. As such, the flash module 69 may be operated by the softwareapplication 65 to trigger at a start of a test and to last for a giventimeframe. Once the flash has been operated, the software application 65may allow the camera module 71 to capture images inside the testingdevice. The software application 65 may choose the camera lens with thehighest quantum efficiency, which may be about 75% at 490 nm. Thesoftware application 65 may capture the amount of light emitted by theAMBs reporters over a given capture time, which may be a fixed time oras long as the emission lasts. If the photons that are captured areabove the threshold specified in the application to eliminate backgroundoptical noise in the system, the pathogen has been detected and this canbe displayed or, via the communications capabilities of the mobile,transmitted. If the photons captured are below the threshold, thenegative results are displayed or communicated, as required.

The smartphone or other mobile device with the ability to induce aresponse of the AMBs fluorophores can be programmed to capture a singleevent (e.g. one flash), or a series of events. Such events then can formdatabases where the results can be deduced by algorithms or AI that isbuilt into the mobile device.

The displaying of the results may be done through the display module 77which the software application 65 may control to display any desiredinformation. As described, the results may be communicated to any othersystem (local or through a wired or wireless connection) or onlinedatabase through the mobile device's connectivity module 75. A copy ofthe data (test result, recorded pictures, name of the user, timestamp,barcode of the testing vial, etc.) may also be locally registered on themobile device's storage module 73. Additionally, when no wired orwireless connections are available, the data may be stored locallybefore being uploaded to an online server once a connection has beenestablished.

Mobile devices may have numerous other sensors which may allow for theregistration and reporting of useful data. As such, the results cancontain personal information (as configured by the user or mandated byan official health organization), such as name, time, date, and GPScoordinates. This may be particularly helpful in conjunction withpandemic tracking applications. The software application 65 may thusprovide an API to connect to such other apps.

Someone skilled in the art will appreciate that the mobile devicecapabilities required to perform the functions listed herein maybeperformed by other computing devices with the same capabilities asdescribed herein.

Aptamer Molecular Beacon Testing Method

As described herein, the aptamer molecular beacon testing device and thesoftware application running on the mobile device may be used to performtesting against a target pathogen. The Aptamer Molecular Beacon may thusbe specific to the target pathogen, such that it may attach to it andits reporter may emit a fluorescent response to a light source. Theability to measure the emitted fluorescence by the mobile device'scamera(s) provides the means to establish the amount of virus found(viral load) inside a fluid sample from a person and thereafter toestablish the strength of the infection (or the lack of infection). Asequence of tests on the same patient over days may further establishthe growth and demise of the infection.

Similarly this method of evaluation of the presence of the virus in anymethod by using the power of a mobile device or similar device canimprove the acquired results and eliminate the human factor such as inPCR based tests for virus or other pathogens.

Now referring to FIG. 8, which is a flowchart of an exemplary method oftesting for a target pathogen using an aptamer molecular beacon testingdevice with a mobile device. The person performing the test, which maybe the test subject or another tester/operator, may start the testingsequence by starting the mobile device's testing application 79. If thetest vials are configured with a barcode, the person performing the testmay thereafter scan the vial's barcode 81. In some embodiments, this mayprovide necessary information to the testing software application, suchas which pathogen is being tested, the state of the vial (e.g.first-time use, reuse, contaminated batch, etc.) and any other relevantinformation.

Afterwards, the tested subject may add a sample of fluid, such as salivaor nasal mucus 83 inside the vial containing the AMBs and a carrierliquid medium. In order to properly mix the sample fluid with the AMBs,the vial may then be shaken 85 for a predetermined amount of time. Oncecompleted, the tester may insert the vial inside the testing device'svial slot 87. The mobile device may further be inserted in the testingdevice's fixture 89 and the test may be subsequently performed 91.

Performing the test 91 may include triggering the flash light source fora given period of time. The image capture may thereafter be done eitherimmediately after the flash light source has extinguished (i.e. whenusing certain fluorophores, they may continue to emit light for a giventime even after the excitation input is removed; this may therefore beused with embodiments of the device which does not include a filter forthe camera as the only light that may be captured would come fromfluorophores) or may be done while the flash light source is stillproviding light to the test vial. In some embodiments, the testingdevice may proceed to a number of flash light source and image capturecycles, such as to have multiple data points that may be used in theimage post-processing step.

Once the test has been completed and the software application has runits analysis of the captured images, the test results may be displayed93 on the mobile device. In some embodiments, the test results may befurther communicated and/or stored locally or on a remote server.

It will be recognized that this method of detecting virus and otherpathogens, forming light emitting molecular beacons formed from aptamerswith fluorophores matched to the flash spectrum of the flash light andspectral sensitivity of the cameras, is a new and efficient means ofdetecting any type of virus, or parts thereof, or surface proteins, thatbinds to the specifically tailored sequences of the probe that is anaptamer or other biological molecular sequence. While aptamers withfluorophore reporters have been described in prior art research, itsuses have generally been limited to detection by spectroscopy or usingspecialized tools and devices. Being able to test and detect a responsefrom a simple mobile device, as a vast majority of the worldwidepopulation have access to, is a significant improvement.

What is claimed is:
 1. A method for detecting a pathogen comprising:adding a test subject sample fluid to a replaceable aptamer molecularbeacon test vial comprising a solution of Aptamer Molecular Beacons anda carrier liquid medium, wherein said Aptamer Molecular Beacons areselected to bind to a protein, RNA or DNA of a target pathogen andfurther comprise a reporter molecule and a quencher molecule, saidreporter molecule being operable to receive light and emit light afterbinding to said target pathogen, said binding modifying a configurationof said Aptamer Molecular Beacons from a quenched configuration, wheresaid quencher molecule is in proximity with said reporter molecule, toan emitting configuration where said quencher molecule and said reportermolecule are separated; mixing a content of said replaceable aptamermolecular beacon test vial; aligning said aptamer molecular beacon testvial with a camera and a flash light source of a mobile computingdevice, wherein said aligning comprises inserting said mobile computingdevice into a slot in a light proof enclosure; using a software testingapplication on said mobile computing device to perform a test to detecta presence of said target pathogen by operating said flash light toilluminate said solution in said vial to excite said reporter moleculeand acquiring at least one image of emission from said reporter moleculeusing said camera; and using said software testing application on saidmobile computing device for at least one of computing an infectionstatus based on said captured image and displaying said infection statuson a display of said mobile computing device.
 2. The method as definedin claim 1, further comprising storing at least one of said capturedimage and said infection status in a storage module of said mobiledevice.
 3. The method as defined in claim 2, further comprising sendingat least one of said captured image and said infection status to aremote server.
 4. The method as defined in claim 1, further comprisingone of receiving a user input code on said mobile computing device andscanning a barcode associated with said vial using said camera of saidmobile computing device to determine at least one of the state of saidreplaceable test vial and a target pathogen being tested.
 5. The methodas defined in claim 4, further comprising using said software testingapplication on said mobile computing device to communicate with a remoteserver to validate said replaceable test vial based on one of said codeand said barcode.
 6. The method as defined in claim 5, furthercomprising preventing said mobile device from being operable todetermine infection status when the state of the replaceable test vialindicates an invalid replaceable test vial.
 7. The method as defined inclaim 4, further comprising preventing said mobile device from beingoperable to determine infection status when the state of the replaceabletest vial indicates an invalid replaceable test vial.
 8. The method asdefined in claim 1, further comprising using said software testingapplication on said mobile computing device to analyze said image toconfirm a presence of said replaceable test vial, wherein a negativeinfection status test result can be prevented.
 9. The method as definedin claim 8, further comprising providing a reference element to beimaged with said vial, wherein said presence is confirmed by processingsaid captured image to identify said reference element.
 10. The methodas defined in claim 1, further comprising placing a bandpass opticalfilter over said camera so as to have sensitivity to emission from saidreporter molecule.
 11. The method as defined in claim 1, wherein saidvial is adapted to have apertures for said flash light source and saidcamera and to provide protection from ambient light.
 12. The method asdefined in claim 1, wherein said acquiring comprises acquiring an imageimmediately following operating said flash light to illuminate saidsolution in said vial to excite said reporter molecule.
 13. The methodas defined in claim 1, wherein said at least one image of emission fromsaid reporter molecule using said camera is analyzed to determine aseverity of an infection or disease.